Identification of differentially expressed peptides in high-throughput proteomics data.

With the advent of high-throughput proteomics, the type and amount of data pose a significant challenge to statistical approaches used to validate current quantitative analysis. Whereas many studies focus on the analysis at the protein level, the analysis of peptide-level data provides insight into changes at the sub-protein level, including splice variants, isoforms and a range of post-translational modifications. Statistical evaluation of liquid chromatography-mass spectrometry/mass spectrometry peptide-based label-free differential data is most commonly performed using a t-test or analysis of variance, often after the application of data imputation to reduce the number of missing values. In high-throughput proteomics, statistical analysis methods and imputation techniques are difficult to evaluate, given the lack of gold standard data sets. Here, we use experimental and resampled data to evaluate the performance of four statistical analysis methods and the added value of imputation, for different numbers of biological replicates. We find that three or four replicates are the minimum requirement for high-throughput data analysis and confident assignment of significant changes. Data imputation does increase sensitivity in some cases, but leads to a much higher actual false discovery rate. Additionally, we find that empirical Bayes method (limma) achieves the highest sensitivity, and we thus recommend its use for performing differential expression analysis at the peptide level.

Proteomic Profiling of Mouse Helper T Cell Differentiation.

Helper T cell differentiation is a key process in the regulation of adaptive immune responses. Here, mouse Th1 and Th2 cells are profiled using high-throughput proteomics to increase the understanding of the molecular biology of Th differentiation to support the design of prophylactic and therapeutic intervention strategies for (infectious) diseases. Protein profiling of Th1/Th2 differentiated cells results in the quantification of almost 6000 proteins of which 41 are differentially expressed at FDR < 0.1, and 19 at the FDR < 0.05 level, respectively. Differential protein expression analysis identifies a number of the expected canonical Th differentiation markers, and gene set analysis using the REACTOME database and a hypergeometric test (FDR < 0.05) confirms that helper T cell pathways are the top sets that are differentially expressed. Additionally, by network analysis, many differentially expressed proteins are associated with the Th1 and Th2 pathways. Data are available via PRIDE database with identifier PXD004532.

Rapid analyses of proteomes and interactomes using an integrated solid-phase extraction-liquid chromatography-MS/MS system.

Here, we explore applications of a LC system using disposable solid-phase extraction (SPE) cartridges and very short LC-MS/MS gradients that allows for rapid analyses in less than 10 min analysis time. The setup consists of an autosampler harboring two sets of 96 STAGE tips that function as precolumns and a short RP analytical column running 6.5 min gradients. This system combines efficiently with several proteomics workflows such as offline prefractionation methods, including 1D gel electrophoresis and strong-cation exchange chromatography. It also enables the analysis of interactomes obtained by affinity purification with an analysis time of approximately 1 h. In a typical shotgun proteomics experiment involving 36 SCX fractions of an AspN digested cell lysate, we detected over 3600 protein groups with an analysis time of less than 5.5 h. This innovative fast LC system reduces proteome analysis time while maintaining sufficient proteomic detail. This has particular relevance for the use of proteomics within a clinical environment, where large sample numbers and fast turnover times are essential.

Proteome adaptation of Saccharomyces cerevisiae to severe calorie restriction in Retentostat cultures.

Stationary-phase, carbon-starved shake-flask cultures of Saccharomyces cerevisiae are popular models for studying eukaryotic chronological aging. However, their nutrient-starved physiological status differs substantially from that of postmitotic metazoan cells. Retentostat cultures offer an attractive alternative model system in which yeast cells, maintained under continuous calorie restriction, hardly divide but retain high metabolic activity and viability for prolonged periods of time. Using TMT labeling and UHPLC-MS/MS, the present study explores the proteome of yeast cultures during transition from exponential growth to near-zero growth in severely calorie-restricted retentostats. This transition elicited protein level changes in 20% of the yeast proteome. Increased abundance of heat shock-related proteins correlated with increased transcript levels of the corresponding genes and was consistent with a strongly increased heat shock tolerance of retentostat-grown cells. A sizable fraction (43%) of the proteins with increased abundance under calorie restriction was involved in oxidative phosphorylation and in various mitochondrial functions that, under the anaerobic, nongrowing conditions used, have a very limited role. Although it may seem surprising that yeast cells confronted with severe calorie restriction invest in the synthesis of proteins that, under those conditions, do not contribute to fitness, these responses may confer metabolic flexibility and thereby a selective advantage in fluctuating natural habitats.

Characterization and usage of the EASY-spray technology as part of an online 2D SCX-RP ultra-high pressure system.

Ultra-high pressure liquid chromatography (UHPLC) systems combined with state-of-the-art mass spectrometers have pushed the limit of deep proteome sequencing to new heights making it possible to identify thousands of proteins in a single LC-MS experiment within a few hours. The recently released EASY-spray technology allows one to implement nano-UHPLC with straightforwardness. In this work we initially characterized the EASY-spray containing a 50 cm column containing <2 µm particles and found that the system allowed 3000 proteins to be identified in 90 minutes. We then asked the question whether a fast and sensitive online 2D SCX-RP UHPLC-MS/MS workflow could compete with 1D long gradient analyses, using total analysis time versus proteome coverage and sample usage as benchmark parameters. The 2D LC-MS strategy consisted of the EASY-spray system that had been augmented by the addition of an SCX column. The conversion was made facile since no additional valves were required and by the use of components containing viper fittings. We benchmarked the system using a human cell lysate digest (<10 µg). The 2D SCX-RP UHPLC-MS/MS workflow allowed the identification of almost 37,000 unique peptides and 6000 proteins in a total analysis time of ~7 hours. On the same system a 1D RP UHPLC-MS/MS workflow plateaued at only 20,000 peptides and 4400 unique proteins and required approx. 8 hours of analysis time. Furthermore, the 2D workflow could continue to increase the proteome coverage with longer analysis times, in fact with a 21 hour analysis we identified 56,600 unique peptides and >7500 proteins. We report, here, that with this fast online SCX-RP UHPLC-MS/MS workflow, the proteome coverage can be substantially extended without significantly compromising the analysis time and sample usage.

Expression of estrogen receptor alpha increases leptin-induced STAT3 activity in breast cancer cells.

Adipositas correlates with an enhanced risk of developing malignant diseases such as breast cancer, endometrial tumor or prostate carcinoma, but the molecular basis for this is not well understood. Potential mechanisms include increased bioavailability of adipocytokines (e.g. leptin) and steroid hormones. Here, we investigated cross-talk between ERalpha (estrogen receptor alpha) and leptin-induced activation of signal transducer and activator of transcription 3 (STAT3), a transactivator of important oncogenes. Upon leptin binding to its receptor Ob-RL (obesity receptor), STAT3 tyrosine phosphorylation and transactivation activity were enhanced by simultaneously expressing ERalpha. Downregulation of ERalpha using small interfering RNA abolished leptin-induced STAT3 phosphorylation. Interestingly, leptin-mediated STAT3 activation was unaffected by co-stimulation with the ERalpha ligands estradiol (E2) or estrogen antagonists ICI182,780 and tamoxifen, implying that enhancement of leptin-mediated STAT3 activity is independent of ERalpha ligands. We also detected ERalpha binding to STAT3 and JAK2 (Janus kinase 2), resulting in enhanced JAK2 activity upstream of STAT3 in response to leptin that might lead to an increased ERalpha-dependent cell viability. Altogether, our results indicate that leptin-induced STAT3 activation acts as a key event in ERalpha-dependent development of malignant diseases.

Differential phosphoproteome profiling reveals a functional role for VASP in Helicobacter pylori-induced cytoskeleton turnover in gastric epithelial cells.

Infection with Helicobacter pylori induces various gastric diseases, including ulceration, gastritis and neoplasia. As H. pylori-induced cellular mechanisms leading to these disease states are widely unclear, we analysed the phosphoproteome of H. pylori-infected gastric epithelial cells. Phosphoproteins from infected cells were enriched using affinity columns and analysed by two-dimensional gel electrophoresis and mass spectrometry. Eleven novel phosphoproteins that showed differentially regulated phosphorylation levels during H. pylori infection were identified. Interestingly, the identified proteins were actin-binding, transport and folding, RNA/DNA-binding or cancer-associated proteins. We analysed functions of one identified H. pylori-regulated candidate, the vasodilator-stimulated phosphoprotein (VASP). H. pylori induced VASP phosphorylation at residues Ser157, Ser239 and Thr278, which was enhanced by the bacterial oncogene cytotoxin-associated gene A. Overexpression of a phosphorylation-resistant VASP mutant efficiently blocked host cell elongation. We identified cGMP-dependent protein kinase G-mediated Ser239 and Thr278 phosphorylation of VASP as a crucial event in H. pylori-dependent host cell elongation. These results suggest that phosphorylated VASP could be a novel target candidate for therapeutic intervention in H. pylori-related gastric diseases.

Phosphoproteomics Reveals Regulatory T Cell-Mediated DEF6 Dephosphorylation That Affects Cytokine Expression in Human Conventional T Cells.

Regulatory T cells (Tregs) control key events of immune tolerance, primarily by suppression of effector T cells. We previously revealed that Tregs rapidly suppress T cell receptor (TCR)-induced calcium store depletion in conventional CD4+CD25- T cells (Tcons) independently of IP3 levels, consequently inhibiting NFAT signaling and effector cytokine expression. Here, we study Treg suppression mechanisms through unbiased phosphoproteomics of primary human Tcons upon TCR stimulation and Treg-mediated suppression, respectively. Tregs induced a state of overall decreased phosphorylation as opposed to TCR stimulation. We discovered novel phosphosites (T595_S597) in the DEF6 (SLAT) protein that were phosphorylated upon TCR stimulation and conversely dephosphorylated upon coculture with Tregs. Mutation of these DEF6 phosphosites abrogated interaction of DEF6 with the IP3 receptor and affected NFAT activation and cytokine transcription in primary Tcons. This novel mechanism and phosphoproteomics data resource may aid in modifying sensitivity of Tcons to Treg-mediated suppression in autoimmune disease or cancer.

Differential gene expression in ERα-positive and ERα-negative breast cancer cells upon leptin stimulation.

In postmenopausal women, adipositas represents a serious risk factor for cancer development and progression. White adipose tissue secretes the 16 kDa hormone leptin which plays a key role in the regulation of appetite and metabolism. An increasing number of reports indicate that leptin also interferes with signal transduction pathways implicated in the development of breast cancer. In our previous study, we identified the estrogen receptor alpha (ERα) as a relevant enhancer of leptin-induced signal transduction leading to transactivation of signal transducer and activator of transcription 3 (Stat3). The purpose of this study is the investigation of specific target gene expression in response to leptin-mediated Stat3 signaling. We performed a comprehensive microarray analysis of ERα-positive and ERα-negative MDA-MB-231 cells upon leptin treatment and identified 49 genes which showed a significant ERα-dependent regulation in leptin-treated MDA-MB-231 cells. There was no intersection with genes which were merely up- or downregulated by ERα expression and only 9 and 11 genes overlapping targets which were regulated by leptin stimulation either in ERα-expressing or ERα-negative MDA-MB-231 cells, respectively. To demonstrate the specificity, expression of three target genes was validated by quantitative real-time PCR. In conclusion, these data imply that leptin can induce a different set of target genes dependent on ERα expression, which might contribute to the development and progression of cancer diseases.